In vivo expression of the nucleolar group I intron-encoded I-dirI homing endonuclease involves the removal of a spliceosomal intron.
The Didymium iridis DiSSU1 intron is located in the nuclear SSU rDNA and has an unusual twin-ribozyme organization. One of the ribozymes (DiGIR2) catalyses intron excision and exon ligation. The other ribozyme (DiGIR1), which along with the endonuclease-encoding I-DirI open reading frame (ORF) is inserted in DiGIR2, carries out hydrolysis at internal processing sites (IPS1 and IPS2) located at its 3' end. Examination of the in vivo expression of DiSSU1 shows that after excision, DiSSU1 is matured further into the I-DirI mRNA by internal DiGIR1-catalysed cleavage upstream of the ORF 5' end, as well as truncation and polyadenylation downstream of the ORF 3' end. A spliceosomal intron, the first to be reported within a group I intron and the rDNA, is removed before the I-DirI mRNA associates with the polysomes. Taken together, our results imply that DiSSU1 uses a unique combination of intron-supplied ribozyme activity and adaptation to the general RNA polymerase II pathway of mRNA expression to allow a protein to be produced from the RNA polymerase I-transcribed rDNA. (+info)
Induction of the tumor suppressor protein p53 restricts cellular proliferation. Since actively growing cells require the ongoing synthesis of ribosomal RNA to sustain cellular biosynthesis, we studied the effect of p53 on ribosomal gene transcription by RNA polymerase I (Pol I). We have measured rDNA transcriptional activity in different cell lines which either lack or overexpress p53 and demonstrate that wild-type but not mutant p53 inhibits cellular pre-rRNA synthesis. Conversely, pre-rRNA levels are elevated both in cells which express mutant p53 and in fibroblasts from p53 knock-out mice. Transient transfection assays with a set of rDNA deletion mutants demonstrate that intergenic spacer sequences are dispensable and the minimal rDNA promoter is sufficient for p53-mediated repression of Pol I transcription. However, in a cell-free transcription system, recombinant p53 does not inhibit rDNA transcription, indicating that p53 does not directly interfere with the basal Pol I transcriptional machinery. Thus, repression of Pol I transcription by p53 may be a consequence of p53-induced growth arrest. (+info)
A new rapidly growing mycobacterial species, Mycobacterium murale sp. nov., isolated from the indoor walls of a children's day care centre.
Scotochromogenic mycobacterial isolates from water-damaged parts of indoor building materials of a children's day care centre represented a phenetically and genetically distinct group of strains. A 16S rDNA dendrogram (1243 bp) showed that the closest species to the new strain MA112/96T was Mycobacterium abscessus. Phylogenetic and phenetic analyses (100 characteristics) grouped the new isolates with M. abscessus, Mycobacterium vaccae, Mycobacterium aurum and Mycobacterium austroafricanum. Ribotyping with Pvull restriction distinguished the 5 isolates from the other 12 most closely related species by the major bands at 6.5-7 kb and 13-15 kb. The cell morphology of the new isolates was typical of mycobacteria, electron microscopy revealed a triple-layered cell wall with an irregular electron-dense outer layer. They grew at 10-37 degrees C, with no growth at 45 degrees C in 5 d. The gene encoding the secreted 32 kDa protein, specific to mycobacteria, was detected by PCR. The main whole-cell fatty acids were characterized by high tuberculostearic acid 10Me-C18:0 (17% at 28 degrees C), which increased with increasing growth temperature (22% at 37 degrees C). The other main fatty acids were C18:1 cis9 and C16:0 (21-20% each), followed by, C17:1 cis9 (14%), C16:1 cis10 (8%) and also a high amount of C20 alcohol (9%). alpha-Mycolic acids, keto-mycolates and wax esters were present (C60-C90), MK-9(H2) (90%) and MK-8(H2) were the main menaquinones. The cellular phospholipids were phosphatidylethanolamine, phosphatidylinositol, phosphatidyl inositolmannosides and diphosphatidylglycerol. Polyamine content was low. G+C content was 72.9 mol%. The new isolates are proposed as a new species, Mycobacterium murale sp. nov. The type strain is MA112/96T (= DSM 44340T). (+info)
Treponema brennaborense sp. nov., a novel spirochaete isolated from a dairy cow suffering from digital dermatitis.
A novel Treponema species was isolated from an ulcerative lesion of a cow suffering from digital dermatitis (DD), a disease which causes painful ulcerations along the coronary band. Among other anaerobic bacteria, high numbers of spirochaetes have been regularly found in DD lesions. Here data are presented of a spirochaete isolated from a DD ulcer. By chemotaxonomy, protein analysis and comparative 16S rDNA sequence analysis this isolate was classified as a treponeme that differed from all Treponema species described previously. The only isolate, DD5/3T, for which the name Treponema brennaborense is proposed, is designated the type strain of the novel species. The strain is a small, highly motile spirochaete that has two periplasmic flagella, one flagellum being attached at each cell pole. Strain DD5/3T exhibits alpha-glucosidase and N-acetyl-beta-glucosaminidase activity and growth is inhibited by rabbit serum. T. brennaborense was phylogenetically most closely related (89.5% 16S rRNA similarity) to Treponema maltophilum, an oral spirochaete isolated from a periodontitis patient. (+info)
RFLP of rRNA genes and sequencing of the 16S-23S rDNA intergenic spacer region of ammonia-oxidizing bacteria: a phylogenetic approach.
It has been established that 16S rRNA gene-based phylogeny gives a low resolution between members of the chemoautotrophic ammonia-oxidizing bacteria (AOB) belonging to the beta-subclass of the Proteobacteria. In this study, 12 isolates of AOB were ribotyped, and the sequences of the 16S-23S rDNA intergenic spacer region (ISR) were determined and used in a phylogenetic study. 16S and 23S rDNA ribotyping revealed that the AOB studied contain only one rrn operon per genome, in contrast to most bacteria, which have 5-10 copies of the rRNA genes per genome. It is likely that the presence of only one set of rRNA genes is related to the slow growth of the AOB. The 16S and 23S rRNA genes of the AOB were shown to be arranged in the classical way: a 16S rRNA gene, an ISR and a 23S rRNA gene. Despite the close phylogenetic relationship among the AOB, the relative location of the rRNA genes in the genome appears to vary considerably. The size of the ISR was approximately 400 bp in the Nitrosomonas isolates and 645-694 bp in the Nitrosospira isolates, suggesting a species-specific size difference in the ISR. The ISR contained two potential tRNA genes in the 5' end in all isolates studied. The similarity values between the ISR sequences of the AOB are low (42.9-96.2%) compared with the 16S rDNA sequence similarity values, and therefore the ISR sequences are valuable as a complementary phylogenetic tool in combination with 16S rRNA gene sequences. The phylogenetic analysis of the AOB based on ISR sequences confirms the 16S rRNA gene-based phylogeny but has the benefit of giving a higher resolution. (+info)
New genus-specific primers for the PCR identification of members of the genera Pseudonocardia and Saccharopolyspora.
Members of the family Pseudonocardiaceae are difficult to identify on the basis of their micromorphology only. The biochemical characterization of each new isolate is a painstaking and time-consuming task which cannot always be undertaken when handling large numbers of strains as is the case in natural product screening programmes. In this study, two sets of genus-specific oligonucleotides were designed which allow rapid detection of members of the genera Pseudonocardia and Saccharopolyspora by means of PCR-specific amplification. The genus specificity of these primers was validated on a wide range of collection strains and the primers were subsequently used to study a group of 106 wild-type isolates that possessed morphological characteristics of the family. Out of this group, 51 strains could be identified as members of the genus Pseudonocardia and only nine isolates could be assigned to the genus Saccharopolyspora. The diversity indicated by whole-cell fatty acid profiles of both wild-type and reference strains was compared with that identified using the oligonucleotide primers. The partial 16S rDNA sequencing of representative wild-type strains was used to validate their genus assignment by PCR-specific amplification. This study shows the industrial usefulness of the application of these direct identification tools as well as the complementary use of two sources of data, PCR-specific amplification results and fatty acid composition, to assess the diversity of a microbial population. (+info)
Reclassification of Brevibacterium oxydans (Chatelain and Second 1966) as Microbacterium oxydans comb. nov.
Phylogenetic and chemotaxonomic analyses indicate that Brevibacterium oxydans is closely related to species of the genus Microbacterium, namely Microbacterium liquefaciens, Microbacterium luteolum and Microbacterium saperdae. DNA-DNA reassociation values of less than 60% between Brevibacterium oxydans and these three Microbacterium species support the distinctness of this misclassified Brevibacterium species, which is reclassified as Microbacterium oxydans comb. nov. (+info)
Reclassification of Brevibacterium incertum (Breed 1953) as Desemzia incerta gen. nov., comb. nov.
Phylogenetic analysis of 16S rDNA indicates that Brevibacterium incertum is not a member of the genus Brevibacterium but related to species of the genus Carnobacterium. Hence, Brevibacterium incertum is not a member of the class Actinobacteria but belongs to the phylogenetically defined broad Bacillus-Lactobacillus cluster. Based upon properties that taxonomically clearly distinguishes Brevibacterium incertum from species of the phylogenetic sister genus Carnobacterium, Brevibacterium incertum is reclassified as Desemzia incerta gen. nov., comb. nov. (+info)